Construction of a recombinant eukaryotic expression. Lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437. Lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following. Generation of variable and fixed length sirna from a novel. Viruses free fulltext potential application of talens. Gibco rpmi 1640 medium with glutamax supplement 61870036 10% gibco fbs a3160401 25 mm gibco hepes 1 m 15630080 1.
Nih3t3 cells also known as 3t3 cells are a standard fibroblast cell line utilized in biological research. At 72 h after transfection, the cells were lysed for 20 min with 0. Invitrogen lipofectamine 3000 transfection reagent l3000008. Does someone have successful transfection in 3t3l1 cells. Transfection protocol lipofectamine 3000 transfection reagent. Protocol for lipofectamine transfection in nih3t3 cells using cells plated in 8well plates 1 mix 0. The 3t3 designation is an abbreviation of 3day transfer, inoculum 3 x 10e5 cells. Transient plasmid dna transfection into hela and hacat cells. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Nih 3t3 mouse embryonic fibroblast cells were initiated from a cell line isolated in 1962 at the new york university school of medicine department of pathology. Xgal staining of nih3t3 24 hours after transfection. Nih3t3 transfection kit swiss mouse fibroblasts altogen.
Transfection protocol lipofectamine 3000 transfection reagent ncih441 lung cancer cells complete growth medium component cat. Usa 800 424 6723 australia 61 2 9914 2800 austria 01 877 89 01. Highest transfection efficiency in many cell types and formats e. To insert plasmid dna into the nih3t3 cells, a lipidmediated plasmid transfection was. Nih3t3 transfection reagent is tested functionally by transfection of nih3t3 cells with a small. Comparison of transfection efficiency between commercialy. Lipofectamine 2000 thermo fisher, xtremegene roche, hiperfect qiagen, and transittko mirus reagents. A the csnk2a1 and csnk2a1p genes were transiently transfected into 293t and nih3t3 cells using lipofectamine 2000 reagent according to manufactures protocol. Prewarm 50ml of optimum stored in cold room at 4c 10min. Xtremegene hp dna transfection reagent highperformance. The mechanism of lipofectamine 2000 mediated transmembrane. Lipofectamine ltx transfection reagent from invitrogen. Induction of reactive oxygen species by bisphenol a and.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Two component lipidbased proprietary formulation optimized for nih3t3 cells transfection. Cell culture, transfection and reporter gene assay nih3t3 or hek293t cells were maintained in dulbeccos modifiedeaglesmediumplus10%fetalbovineserum. This reference provides general guidelines and an optimized procedure to transfect stealth rnai or sirna into mouse 3t3 l1 fibroblast cells atcc, cat. In biontex experiments gfp positivity increased to 90. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Optimized conditions for successful transfection of human. Nih3t3 cells affected cell survival and proliferation in a much. B hela cells were transfected with luciferase encoding plasmid dna using either transit. Invitrogen lipofectamine 2000 transfection reagent fisher. Nanomagnetic gene transfection for nonviral gene delivery in nih 3t3 mouse embryonic fibroblasts. Genscript recommends using lipofectamine 2000 for all transfections. Nih3t3 met m1268t cells were transfected using standard reverse transfection protocols with lipofectamine. Lipofectamine 2000 invitrogen reagent was used for transfection.
Lipofectamine transfection reagent is a trusted, value reagent with many years of cited use in thousands of publications and with many cell lines. Cell transfectionthe establishment of inducible shrna stable cell lines has been described previously 18. Have you even transfected nih3t3 fibroblasts using pei reagent. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Dysregulated phosphorylation of rab gtpases by lrrk2. Genomic rna from the 2019ncovhong kongvm200010612020 strain and a synthetic rna preparation of the sarscov 2003 strain. Nih3t3 cells were either treated with both lipofectamine and specific talen plasmids mcmv12, 34 and 56, nonspecific controls w1fsw7r1 and kshv12 or only lipofectamine as a. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Nih3t3 cells line is a good transfection host susceptible to sarcoma and leukemia viruses. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Add 6l of lipofectamine2000 from biostores and 100 l of. Life science group bulletin 5969 rev a useg 100503 0310 sig 1109 biorad laboratories, inc. Plasmids were transfected using lipofectamine plus reagent invitrogenaccordingtothemanufacturersinstructions.
My colleague has a lot of experience transfecting nih3t3. Sep 25, 2007 invitrogen have added a new product, lipofectamine ltx, to their extensive range of transfection products. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Invitrogen lipofectamine 3000 transfection reagent fisher. Metafectene pro and lipofectamine had the highest efficiencies. Xtremegene hp dna transfection reagent is a multicomponent reagent that forms a complex with dna, then transports the complex into animal or insect cells. Comparative transfection efficiency of jetprime versus its main competitor.
Transfection of nih3t3 ce lls with lipofectamine 2000. Lipofectamine transfection of c2c12 cells bridges lab. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. The same procedure was also applied to determine egfp expression in d1 dendritic cells, raw 246. Use a transfection control dsirna to visually monitor transfection efficiency. Ampkdependent activation of the cyclin ycdk16 complex. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Conditions were used according to the manufacturers recommendation for both lipofectamine. What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual which addresses these issues and helps tremendously cut down the initial time of protocol optimization. Using nih3t3 fibroblasts, cells were grown to 7090% confluence in a 6well plastic cell culture dish. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. Because of the low infection efficiency, i am a bit worried the same thing will happen on transient transfection using lipofectamine 2000 on nih3t3.
Human hek293 and mouse nih3t3 cells were seeded 24 h before transfection. Protocol for lipofectamine transfection in nih3t3 cells. Transfection efficiency of php was examined according to the protocol depicted above. Cotransfection of gk and mhpins genes into hepg2 cells.
In hacat cells, lipofectamine 2000 achieved a transfection efficiency of 22. Lipofectamine transfection reagent is one of our firstgeneration, cationiclipid transfection reagents formulated for the transfection of dna into eukaryotic cells. Lipofectamine 2000 transfection reagent from invitrogen. Laura schmidt nci, frederick, md, usa and maintained as previously described. Introducing the monster green fluorescent protein phmgfp vector using the transfast transfection reagent and the optimized transfection conditions resulted in the cell notes issue 7 2003 14 neuroscience transfecting a human neuroblastoma cell line with monster green fluorescent protein by natalie betz, ph. C, d neuro2a and gc1 cells in 96well plates were transfected with 16 and 10 ng of pcdna3fdj1 or pcdna3f, respectively, using lipofectamine 2000. Bgalactosidase expression was measured 48 hours posttransfection. Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates. Nih3t3 is commonly used for in vitro studies, so it gives me the perception its easy to transfect and infect. Transfection of nih3t3 cells and screening of stable expressing cell lines nih3t3 atcc cells were transfected with pcdna3. Nih3t3 cells stably expressing metactivating mutation m1268t were provided by dr.
Dna transfection was performed with lipofectamine 2000 invitrogen, carsbad, ca or fugene 6 promega, madison, wi. Microrna105 suppresses cell proliferation and inhibits. Mar 27, 2007 too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. Lipofectamine 3000 reagent thermo fisher scientific kr. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. Transfection was measured by luciferase activity using a conventional assay. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. The 3t3 cell line was originally established from a swiss albino mouse embryo tissue in 1962 by two scientists at new york university school of medicine. Pik3ca hotspot mutations differentially impact responses. I use 24well plates for transfections in triplicates. Chok1, cos7, hela, hepg2, huh7, huvec, imr90, mcf7, mef, and nih3t3.
Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. For additional information on scaling your transfection reaction, see page 4. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines. Cells were transfected with a firefly luciferase encoding plasmid dna 0. Scaling up or down lipofectamine 3000 reagent transfections. I used lipofectamine 2000, 3000, pei, fugene 6 and hd, electroporation and no transfected cell.
This reference provides a recommended procedure to transfect plasmid dna into nih3t3 mouse embryonic fibroblasts atcc cat. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from a difficult to transfect cell line that had been giving us trouble with the lipofectamine 2000. Nih3t3 cell line transfection, expression and culture. Tissue culture and transfection assays the h9c2 cells, nih3t3 cells and neonatal rat cardiac myocytes were cultured as previously described 1,14. Nih3t3 transfection reagent is supplied in liquid form at a concentration of 0. Using this protocol, the immortal cell line begins to thrive and stabilize in cell culture. Add 6l of lipofectamine2000 from biostores and 100 l of optimum to. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Myctagged lrrk2 and v5tagged rabs rab1a, 3c, 8a, or 35 were cotransfected with lipofectamine 2000 into hek293 cells. Transfection protocol lipofectamine 3000 transfection reagent ncih1299 lung cancer cells complete growth medium component cat. Jul 24, 2007 lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol.
Platinume retroviral packaging cell line, ecotropic. Metafectene pro had less toxicity than lipofectamine. Altogen biosystems offers lipidbased transfection reagent kits for the nih3t3 cell line that yield high transfection efficiency, as detected by qrtpcr. Dna complexes with commercial transfection reagents lipofectamine 2000. Universal electroporation buffer is provided to increase cell transfection efficiency. Nih3t3 cells were transfected with plasmids by lipofectamine 2000. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Cell culture, transfection and cdna library screening. Using four different transfection reagents fugene hd, lipofectamine 2000, effectene, and lipofectamine ltx with plus reagent, jacobsen et al. Transfecting a human neuroblastoma cell line with monster. Cells in a 6 well dish, plated and at 70% confluence. Hepatocellular carcinoma hcc, which accounts for 8090% of primary liver cancers, is the third most common cause of cancerrelated mortality worldwide.
Lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells and provides the following advantages. Nih3t3 cells transfected for 2 hours with 6 l transpass p protein transfection reagent and 1 g igg labeled with a fluorescent dye a 549 fluorophore. Pdf nanomagnetic gene transfection for nonviral gene. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment.
Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. A novel protein tyrosine kinase nok that shares homology. Lipofectamine 2000 reagent thermo fisher scientific. Fluorescent protein exhibited persistent expression in nih3t3 cells over a 7day period, again suggesting the nontoxic nature. How to increase transfection efficiency when lipofectin 2000. Transfection protocol lipofectamine 3000 transfection reagent ncih292 lung cancer cells complete growth medium component cat.
Luciferase activity was measured 24 h after transfection. Transfecting stealth rnai or sirna into 3t3 l1 cells. The cells were fixed, dapi stained and photographed 24 hours posttransfection. Transfection protocol library the transfection protocol online library contains protocols obtained from the. A highly efficient method of transfecting nih3t3 cells. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. A sample protocol is listed here for transfection experiments performed in 6well plates. Lipofectamine 2000 reagent is a proprietary formulation that facilitates highly efficient delivery of stealth rnai or short interfering rna sirna to mammalian cells for rnai analysis. It can be used for transfection of adherent or suspension cells with dna or rna including. However, the lipo 2000 transfection efficiency is low about 5% to 15% acting on the cell line 3t3 or neuronal cell line gt17 compared to the cell line 293at about 80% to 90%. On the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24well plate using lipofectamine 3000 transfection reagent. Other cell lines may be refractory to some or most of these. Much better transfection efficiency with all reagents. Identificationofzincfingerbeddomaincontaining3 zbed3.